CD4+ T cells (T/CD4+), which play a key role in most protective immune responses, are triggered by peptide-MHC class II complexes that are formed within endosomal compartments and subsequently transported to the surfaces of antigen-presenting cells (APC). While many details of class II-restricted processing and presentation have been elucidated, a number of key questions remain. What are the functional differences between distinct APC types and what are the bases for such differences? What mechanisms underlie the acquisition of epitope by MHC class II molecules? How is processing and presentation of exogenously provided antigen different from that of endogenously produced antigen? For many years we have utilized in vitro and in vivo murine systems to ask questions along these lines, with the A/PR/8/34 influenza virus for our antigen system. Besides its relevance to human health, flu has several remarkable properties that provide considerable advantages for such studies. In this research plan, we will continue to exploit the biology of influenza, in particular its glycoproteins HA and neuraminidase (NA) to extend several areas of fruitful research from the previous funding period and to introduce several new areas of investigation. With a focus on two epitopes from HA and one epitope from NA, all presented by the same class II molecule (IEd) we propose to: 1) test the hypothesis that structural context, rather than affinity for class II, is the basis for the remarkably different presentation phenotypes of the two HA epitopes, 2) determine the role of the GILT thiol reductase in the generation of the 3 epitopes, 3) explore the role of the proteasome in the class II restricted processing of endogenously-produced antigens, 4) test the notion that epitope-loaded recycling class II molecules take two routes to the cell surface, one direct and the other by way of the late endosome, and 5) determine how the H2-M regulator H2-O influences the expression of the epitopes. Woven into most of these pursuits will be a comparison of APC types, particularly dendritic cells (DCs) vs. other cell types, since DCs have a strikingly different capacity for presentation of the three epitopes. A key goal of these studies, besides contributing at a fundamental, mechanistic level, is to elucidate how, through structure and function, "relevant" antigens such a flu differ in their processing and presentation from "shelf" antigens that have been traditionally used, with the notion that understanding such differences may be crucial for optimizing immunity to influenza and similar viruses.